Tuberculosis (TB) is a devastating disease of global proportions. The causative agent of this disease, Mycobacterium tuberculosis (Mtb), latently infects approximately a third of the world's population and causes nearly two million deaths a year. Additionally, deadly synergy with HIV-infection and the emergence of drug resistant strains have further complicated efforts to treat and manage the disease. Understanding the signaling pathways that allow Mtb to respond to changing host environments will likely facilitate the identification of desperately needed new treatment strategies. Adenosine 3',5'-cyclic monophosphate (cAMP) is a key signaling molecule in Mtb gene regulation. Mtb possesses 15 adenylyl cyclases (ACs), many of which respond to different environmental stimuli in vitro. This large repertoire of Mtb ACs raises the issue of whether these ACs function in distinct signaling pathways in vivo. How such specificity could be achieved when ACs all increase cAMP levels is a major unresolved question. This application tests the hypothesis that specificity exists in Mtb cAMP signaling. Additionally, this application tests whether Mtb ACs differentially interact with and activate either of the two recently identified cAMP receptor protein-like transcription factors (TFs). I propose to determine whether ACs function in distinct signaling pathways that do not completely overlap using a proteomics approach on AC deletion strains grown under high nutrient, neutral pH conditions. I will also assess physical proximity between specific ACs and the two TFs, using mycobacterial protein fragment complementation (M-PFC), coimmunoprecipitation, and indirect immunofluorescence microscopy. If specificity in cAMP signaling is occurring via physical proximity, I expect that specific ACs will bind to or localize with one TF, but not the other. I will also characterize the cAMP signaling pathway by identifying genes (using semi-quantitative reverse transcription polymerase chain reaction and chromatin immunoprecipitation) that are commonly regulated at the transcriptional level by a specific AC and either of the two TFs. Results from these studies will provide critical new information on the role of specificity in Mtb cAMP signaling. Such knowledge could identify new Mtb drug targets or treatment strategies. PUBLIC HEALTH RELEVANCE: Approximately two million people die each year from infection with Mtb. The identification of new Mtb drug targets and improved treatment strategies are sorely needed to control this emerging epidemic.